(a) Field of the Invention
The present invention relates to methods for detecting the effect of test samples on UDP-glucose:glycoprotein glucosyltransferase (UGGT) activity. The present invention also relates to the nucleic acid encoding for mammalian UGGT and to recombinant mammalian UGGT.
(b) Description of the Prior Art
The quality control system of the endoplasmic reticulum (ER) ensures that only folded proteins proceed further along the secretory pathway. Some of the abundant ER proteins are components of molecular chaperone systems which bind to unfolded proteins, retaining them in the ER. The folding enzymes are also abundant in the ER and comprise several disulfide isomerases and prolyl peptidyl isomerases (1, 2). How chaperones and folding enzymes interact to facilitate protein folding in the ER is not known.
Calnexin and calreticulin participate in a molecular chaperone system which integrates the processes of N-glycosylation and quality control (3, 4). They both are lectins that bind N-glycans of the form GlcNAc2Man9Glc1 which result from the removal of the two outer glucoses from GlcNAc2Man9Glc3 oligosaccharides by the sequential action of glucosidases I and II. Removal of the last glucose by glucosidase II prevents binding by calnexin and calreticulin. Then, if the glycoprotein is unfolded, a glucose residue is added back to the high mannose core by the enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) which recognizes unfolded proteins (5). Consequently, during folding glycoproteins undergo cycles of binding and release from calnexin and calreticulin which are driven by the addition and removal of a glucose residue (4). As a result of the specificity of UGGT, only unfolded glycoproteins bind to calnexin and calreticulin in vivo (6, 7), even though these lectins do not recognize the conformation of their protein substrates (8, 9).
Monoglucosylated glycoproteins, in addition to binding to calnexin and calreticulin, can also be cross-linked to the ER protein ERp57 (10, 11) (also known as ER-60, ERp60, ERp61, GRP58, P58, HIP-70 or Q-2; ref. (11) and refs therein). ERp57 is homologous to protein disulfide isomerase (PDI) and has been shown to exhibit thiol-disulfide oxidoreductase activity in vitro (12).
UDP-glucose glycoprotein:glucosyltransferase (UGGT) is a soluble enzyme of the endoplasmic reticulum (ER) which catalyzes the addition of a glucose residue onto asparagine-linked oligosaccharides of the form [GlcNAc]2-(Man)7-9 which are present on incorrectly folded glycoproteins (Parodi, 1984; Trombetta, 1989; Sousa, 1992). UGGT is now thought to be responsible for the prolonged binding of unfolded glycoproteins to the ER lectins calnexin and calreticulin (Ou, 1993; Peterson, 1995), and is therefore a key component of the quality control system of the ER which ensures that only correctly folded and assembled protein are exported.
N-linked glycans are synthesized as a dolichol-anchored unit of 14 residues [GlcNAc]2-(Man)9-(Glc)3 (Herscovics, 1993). After transfer of the oligosaccharide to a protein in the ER, the terminal glucose is removed by glucosidase I (Bause, 1986). Then, glucosidase II successively removes the two remaining glucose residues (Hubbard, 1981) leaving the [GlcNAc]2-(Man)9 core oligosaccharide. If the glycoprotein is not correctly folded, the innermost glucose is added back by UGGT, which can discriminate between folded and unfolded substrates (Trombetta, 1989; Sousa, 1995). The known specificity of UGGT for unfolded proteins and the in vivo abrogation of binding to calnexin by inhibitors of the two glucosidases led to the formulation of a model whereby only monoglucosylated glycoproteins bind to calnexin or calreticulin (Hammond, 1994; Helenius, 1997). Unfolded glycoproteins are thought to undergo cycles of binding to and release from calnexin and calreticulin. Glucosidase II does not recognize the conformation of the polypeptide but removes indiscriminately the glucose which is present (Rodan, 1996). If the glycoprotein is folded, it is not reglucosylated by UGGT and escapes from the cycle. If the glycoprotein is still unfolded, it is reglucosylated and remains trapped in the cycle (Hxc3xa9bert, 1995). The ensemble of UGGT, glucosidase II, calnexin and calreticulin can be considered as a molecular chaperone system as their interplay results in the binding and release of unfolded proteins. The binding of unfolded glycoproteins to calnexin and calreticulin relies on the specificity of UGGT for unfolded substrates, as both lectins were shown not to recognize the conformation of their substrates (Rodan, 1996; Zapun, 1997).
UGGT has been previously purified to homogeneity from rat liver and shown to have an apparent molecular weight of about 150 kDa on denaturing gels and 270 kDa in native conditions (Trombetta, 1992). This enzyme catalyzes the transfer of the glucose residue from UDP-glucose onto the distal mannose residue of the longest branch of the core oligosaccharide in an xcex1-1,3 linkage (Trombetta, 1989). [GlcNAc]2-(Man)9 was found to be a better acceptor for the glucose transfer than [GlcNAc]2-(Man)8, which in turn is better than [GlcNAc]2-(Man)7 (Sousa, 1992). Oligosaccharides with a lower mannose content are not substrates of UGGT. Most importantly, the glucosylation reaction is far more efficient if the glycoprotein substrate is unfolded. The effect of denaturation is not to expose the oligosaccharides but to make protein determinants required for enzymatic activity accessible to UGGT (Sousa, 1992). The enzyme was also shown to have some affinity for hydrophobic peptides (Sousa, 1995) and unfolded proteins are known to expose hydrophobic residues that are normally buried in the folded conformation. Unfolded glycoproteins treated with endo-xcex2-N-acetylglucosaminidase H (EndoH) were found to be competitive inhibitors of the reaction whereas denatured non-glycosylated proteins were not. This finding indicated that the innermost N-acetylglucosamine residue, which remains attached to the denatured polypeptide after treatment with EndoH, is presumably required for substrate recognition (Sousa, 1992).
The cDNA encoding UGGT from Drosophila melanogaster has been cloned (Parker, 1995; Accession #U20554) as well as from the fission yeast Schizosaccharomyces pombe, GPT1 (Fernxc3xa1ndez, 1996; Accession # U38417). The sequence of the gene encoding UGGT from Ceanorabditis elegans is also available as a result of the genome sequencing project of this organism (Wilson, 1994; Accession #U28735). These genes all encode proteins of about 1500 amino acids with a N-terminal signal sequence and a C-terminal retention signal, as expected for ER luminal proteins. The gene for UGGT is not essential in S. pombe and no apparent phenotype can be observed upon its disruption (Fernxc3xa1ndez, 1996).
One of the major hurdle in the study of UGGT is the scarcity of appropriate substrates for detailed in vitro studies. High mannose oligosaccharides are transient species in the cell as they are further modified along the secretory pathway following their exit from the ER. Most secreted glycoproteins do not contain the [GlcNAc]2-(Man)7-9 acceptor glycans and when they do, the appropriate oligosaccharides constitute only a small fraction of the total, as it is the case for bovine pancreatic ribonuclease B (RNase B) (Rudd, 1994; Zapun, 1997). As the pathways of oligosaccharide biosynthesis in Saccharomyces cerevisiae are well characterized, this organism provides a mean to produce various forms of glycans by genetic engineering.
In yeast, the transfer of N-linked oligosaccharides onto proteins is analogous to that in mammals. However, after glucose trimming, the remaining [GIcNAc]2-(Man)9 core oligosaccharide is modified differently (Herscovics, 1993). One of the terminal mannose residues is removed by the action of an ER xcex1-mannosidase, product of the gene MNS1 (Jelinek-Kelly, 1988). The remaining terminal mannose units are the acceptors for the addition of further mannose residues by an xcex1-1,3-mannosyltransferase encoded by the gene MNN1 (Graham, 1992). Finally, one of the GlcNAc residue is also the site of attachement of an additional mannose residue by an xcex1-1,6-mannosyltransferase encoded by the OCH1 gene (Nakayama, 1992). A long polysaccharide chain is then built onto this additional mannose residue to produce the typically hyperglycosylated yeast proteins. A yeast strain having these three genes disrupted is expected to produce glycoproteins which have only [GlcNAc]2-(Man)9 oligosaccharides.
It is an aim of the present invention to provide a method for determining UGGT activity.
It is also an aim of the present invention to provide an isolated nucleic acid comprising a nucleotide sequence encoding for a mammalian UGGT.
It is also an aim of the present invention to provide a recombinant mammalian UGGT.
In accordance with the present invention there is provided a method for determining the effect of a test sample on UGGT activity which comprises the steps of:
a) exposing a UGGT substrate to a labeled donor in the presence of the test sample and UGGT; and
b) detecting the amount of labeled donor which was transferred to the UGGT substrate wherein an increase of donor intake when compared to a control means that the test sample is a UGGT stimulator and a decrease means that the test sample is a UGGT inhibitor.
In accordance with the present invention, there is provided an isolated nucleic acid comprising a nucleotide sequence or an analogue thereof provided that the nucleotide sequence or an analogue thereof encodes for mammalian UGGT.